The erythropoietin (Epo) receptor is a model for understanding signal transduction mechanisms of the cytokine receptor superfamily. Calcium signaling plays an important role in cell proliferation and apoptosis, yet little is known about regulation of ion channels by hematopoietic growth factors. Using quantitative fluorescence microscopy and electrophysiology, our laboratory was the first to demonstrate that Epo modulates the intracellular calcium concentration ([Ca2+] 0 through activation of members of the TRPC ion channel family. The transient receptor potential (TRP) superfamily is a diverse group of voltage-independent calcium permeable channels which are involved in sustained calcium entry in nonexcitable cells. TRPC3 and TRPC6 are members of the TRPC subfamily which are expressed in human erythroid cells. TRPC3 is activated by Epo, while TRPC6 inhibits TRPC3 activation. TRPC2 is also activated by Epo in murine erythroid cells but is a pseudogene in humans. In this grant, Specific Aim 1 will study the mechanisms by which Epo regulates [Ca2*1i through activation of TRPC3. We will examine (1) mechanisms of TRPC3 tyrosine phosphorylation after Epo stimulation by identifying the involved kinase and key tyrosine residues on TRPC3 which are phosphorylated and required for channel activation; (2) the role of phospholipase C and IP3R in TRPC3 activation by Epo; and (3) the mechanisms through which Epo regulates TRPC3 cell surface localization. Specific Aim 2 will examine the mechanisms through which TRPC6 inhibits TRPC3 activation by Epo. TRPC3 and TRPC6 expression are regulated during normal erythroid differentiation. We will determine whether TRPC6 inhibits TRPC3 membrane localization or tyrosine phosphorylation and identify channel domains which are responsible for the different responses to Epo. Specific Aim 3 will examine the functional importance of TRPC3 in ervthropoiesis using TRPC3 whole animal and tissue specific knockout mice and TRPC2-/-TRPC3-/- double knockouts. Preliminary results show that TRPC2 knockout animals are protected from hemolysis in response to oxidative stress. Understanding regulation of TRP channels by hematopoietic growth factors including erythropoietin is of fundamental biological importance, and the knowledge gained from these studies will be widely applicable to regulation of hematopoiesis by other members of the cytokine receptor superfamily.